T helper cell epitopes

ABSTRACT

The present invention provides T helper cell epitopes and compositions for use in inducing an immune response comprising at least one of these epitopes. The epitopes are contained within a peptide sequence selected from the group consisting of SSKTQTHTQQDRPPQPS (SEQ ID NO: 1); QPSTELEETRTSRARHS (SEQ ID NO: 2); QSLRTSLEQSNKAIEEI (SEQ ID NO: 18); and DESSCVFVSESAICSQN (SEQ ID NO: 23).

RELATED APPLICATION DATA

This application is a divisional of application Ser. No. 10/705,819,filed Nov. 13, 2003, which is a divisional of application Ser. No.09/890,650, filed Mar. 22, 2002, now U.S. Pat. No. 6,685,947, which is a371 of International Application No. PCT/AU00/00070, filed Feb. 7, 2000,the contents of which are hereby incorporated by reference in thisapplication.

This application is a divisional patent application of U.S. Ser. No.10/705,819 filed on Nov. 13, 2003, which is a divisional patentapplication of U.S. Ser. No. 09/890,650 filed Mar. 22, 2002 and nowissued as U.S. Pat. No. 6,685,947, which is a 371 of InternationalPatent Application No. PCT/AU00/00070 filed on Feb. 7, 2000, whichclaims benefit of foreign priority under 35 USC § 119 from AustralianPatent Application No. PP8533 filed on Feb. 5, 1999 and AustralianPatent Application No. PQ2013 filed on Aug. 4, 1999. The contents ofU.S. Ser. No. 10/705,819 filed on Nov. 13, 2003 and U.S. Ser. No.09/890,650 (U.S. Pat. No. 6,685,947) filed Mar. 22, 2002 are eachincorporated in their entirety by reference.

FIELD OF THE INVENTION

The present invention relates to T helper cell epitopes derived fromCanine Distemper Virus (CDV). The present invention relates tocompositions including at least one T helper cell epitope and optionallyB cell epitopes and/or CTL epitopes.

BACKGROUND OF THE INVENTION

For any peptide to be able to induce an effective antibody response itmust contain particular sequences of amino acids known as epitopes thatare recognised by the immune system. In particular, for antibodyresponses, epitopes need to be recognised by specific immunoglobulin(Ig) receptors present on the surface of B lymphocytes. It is thesecells which ultimately differentiate into plasma cells capable ofproducing antibody specific for that epitope. In addition to these Bcell epitopes, the immunogen must also contain epitopes that arepresented by antigen presenting cells (APC) to specific receptorspresent on helper T lymphocytes, the cells which are necessary toprovide the signals required for the B cells to differentiate intoantibody producing cells.

In the case of viral infections and in many cases of cancer, antibody isof limited benefit in recovery and the immune system responds withcytotoxic T cells (CTL) which are able to kill the virus-infected orcancer cell. Like helper T cells, CTL are first activated by interactionwith APC bearing their specific peptide epitope presented on thesurface, this time in association with MHC class I rather than class IImolecules. Once activated the CTL can engage a target cell bearing thesame peptide/class I complex and cause its lysis. It is also becomingapparent that helper T cells play a role in this process; before the APCis capable of activating the CTL it must first receive signals from thehelper T cell to upregulate the expression of the necessarycostimulatory molecules.

Helper T cell epitopes are bound by molecules present on the surface ofAPCs that are coded by class II genes of the major histocompatibilitycomplex (MHC). The complex of the class II molecule and peptide epitopeis then recognised by specific T-cell receptors (TCR) on the surface ofT helper lymphocytes. In this way the T cell, presented with anantigenic epitope in the context of an MHC molecule, can be activatedand provide the necessary signals for the B lymphocyte to differentiate.Traditionally the source of helper T cell epitopes for a peptideimmunogen is a carrier protein to which peptides are covalently coupledbut this coupling procedure can introduce other problems such asmodification of the antigenic determinant during the coupling processand the induction of antibodies against the carrier at the expense ofantibodies which are directed toward the peptide (Schutze, M. P.,Leclerc, C. Jolivet, M. Audibert, F. Chedid, L. Carrier-induced epitopicsuppression, a major issue for future synthetic vaccines. J. Immunol.1985, 135, 2319–2322; DiJohn, D., Torrese, J. R. Murillo, J. Herrington,D. A. et al. Effect of priming with carrier on response to conjugatevaccine. The Lancet. 1989, 2, 1415–1416). Furthermore, the use ofirrelevant proteins in the preparation introduces issues of qualitycontrol. The choice of appropriate carrier proteins is very important indesigning peptide vaccines and their selection is limited by factorssuch as toxicity and feasibility of their large scale production. Thereare other limitations to this approach including the size of the peptideload that can be coupled and the dose of carrier that can be safelyadministered (Audibert, F. a. C., L. 1984. Modern approaches tovaccines. Molecular and chemical basis of virus virulence andimmunogenicity., Cold Spring Harbor Laboratory, New York.). Althoughcarrier molecules allow the induction of a strong immune response theyare also associated with undesirable effects such as suppression of theanti-peptide antibody response (Herzenberg, L. A. and Tokuhisa, T. 1980.Carrier-priming leads to hapten-specific suppression. Nature 285:664;Schutze, M. P., Leclerc, C., Jolivet, M., Audibert, F., and Chedid, L.1985. Carrier-induced epitopic suppression, a major issue for futuresynthetic vaccines. J Immunol 135:2319; Etlinger, H. M., Felix, A. M.,Gillessen, D., Heimer, E. P., Just, M., Pink, J. R., Sinigaglia, F.,Sturchler, D., Takacs, B., Trzeciak, A., and et, a. 1988. Assessment inhumans of a synthetic peptide-based vaccine against the sporozoite stageof the human malaria parasite, Plasmodium falciparum. J Immunol140:626).

In general then, an immunogen must contain epitopes capable of beingrecognised by helper T cells in addition to the epitopes that will berecognised by surface Ig or by the receptors present on cytotoxic Tcells. It should be realised that these types of epitopes may be verydifferent. For B cell epitopes, conformation is important as the B cellreceptor binds directly to the native immunogen. In contrast, epitopesrecognised by T cells are not dependent on conformational integrity ofthe epitope and consist of short sequences of approximately nine aminoacids for CTL and slightly longer sequences, with less restriction onlength, for helper T cells. The only requirements for these epitopes arethat they can be accommodated in the binding cleft of the class I orclass II molecule respectively and that the complex is then able toengage the T-cell receptor. The class II molecule's binding site is openat both ends allowing a much greater variation in the length of thepeptides bound (Brown, J. H., T. S. Jardetzky, J. C. Gorga, L. J. Stem,R. G. Urban, J. L. Strominger and D. C. Wiley. 1993. Three-dimensionalstructure of the human class II histocompatibility antigen HLA-DR1.Nature 364:33) with epitopes as short as 8 amino acid residues beingreported (Fahrer, A. M., Geysen, H. M., White, D. O., Jackson, D. C. andBrown, L. E. Analysis of the requirements for class II-restricted T-cellrecognition of a single determinant reveals considerable diversity inthe T-cell response and degeneracy of peptide binding to I-Ed J.Immunol. 1995. 155: 2849–2857).

Canine distemper virus (CDV) belongs to the subgroup of morbillivirus ofparamyxovirus family of negative-stranded RNA viruses. Other viruseswhich are members of this group are measles virus and rinderpest virus.Development of peptide based vaccines has aroused considerable interestin identification of B and T cell epitopes from sequences of proteins.The rationale for using T cell epitopes from proteins such as the Fprotein of CDV is that young dogs are inoculated against CDV in earlylife and will therefore possess helper T cells specific for helper Tcell epitopes present on this protein. Subsequent exposure to a vaccinewhich contains one or more of the epitopes will therefore result inrecruitment of existing helper T cells and consequently an enhancedimmune response. Such helper T cell epitopes could, however, beadministered to unprimed animals and still induce an immune response.The present inventors aimed to identify canine T cell epitopes from thesequence of CDV fusion protein so that these epitopes can then be usedin the design of peptide based vaccines, in particular, for the canineand related species.

LHRH (Luteinising hormone releasing hormone) is a ten amino acids longpeptide hormone whose sequence is conserved in mammals. It is secretedby the hypothalamus and controls the reproductive physiology of bothmales and females. The principle of development of LHRH-basedimmunocontraceptive vaccines is based on observations that antibodies toLHRH block the action of the hormone on pituitary secretion ofluteinising hormone and follicle stimulating hormone, leading to gonadalatrophy and sterility in mammals.

Most LHRH vaccines that have been developed consist of LHRH chemicallyconjugated to protein carriers to provide T cell help for the generationof anti-LHRH antibodies. It has been shown that upon repeatedinoculation of LHRH-protein carrier conjugates the anti-LHRH titredecreases due to the phenomenon known as “carrier induced epitopesuppression”. One aim of the present inventors is to replace proteincarriers in the vaccines with defined T helper epitopes (TH-epitopes) soas to eliminate “carrier induced epitope suppression”.

SUMMARY OF THE INVENTION

The present inventors have identified a number of 17 residue peptideseach of which includes a T helper cell epitope. As will be readilyappreciated the majority of these peptides are not minimal T helper cellepitopes. Typically class II molecules have been shown to be associatedwith peptides as short as 8 amino acids (Fahrer et al., 1995 ibid) butusually of 12–19 amino acids (Chicz, R. M., Urban, R. G., Gorga, J. C.,Vignali, D. A. A., Lane, W. S. and Strominger, J. L. Specificity andpromiscuity among naturally processed peptides bound to HLA-DR alleles.J Exp Med 1993, 178, 27–47; Chicz, R. M., Urban, R. G., Lane, W. S.,Gorga, J. C., Stem, L. J., Vignali, D. A. A. and Strominger, J. L.Predominant naturally processed peptides bound to HLA-DR1 are derivedfrom MHC-related molecules and are heterogeneous in size. Nature 1992,358, 764–8), although, peptides up to 25 amino acids in length have beenreported to bind to class II (reviewed in Rammensee, H. -G. Chemistry ofpeptide associated with class I and class II molecules. Curr OpinImmunol 1995, 7, 85–95.).

Thus peptide epitopes that range in length between 8 and 25 amino acidresidues can bind to class II molecules. The shorter peptides are “core”epitopes that may have less activity than longer sequences but it is atrivial exercise to truncate longer sequences at the N- or theC-terminus to yield shorter sequences that have the same or betteractivity than the parent sequence.

Accordingly in a first aspect the present invention consists in a Thelper cell epitope, the epitope being contained within a peptidesequence selected from the group consisting of SSKTQTHTQQDRPPQPS (SEQ IDNO: 1); QPSTELEETRTSRARHS (SEQ ID NO: 2); RHSTTSAQRSTHYDPRT (SEQ ID NO:3); PRTSDRPVSYTMNRTRS (SEQ ID NO: 4); TRSRKQTSHRLKNIPVH (SEQ ID NO: 5);SHQYLVIKLIPNASLIE (SEQ ID NO: 6); IGTDNVHYKIMTRPSHQ (SEQ ID NO: 7);YKIMTRPSHQYLVIKLI (SEQ ID NO: 8); KLIPNASLIENCTKAEL (SEQ ID NO: 9);AELGEYEKLLNSVLEPI (SEQ ID NO: 10); KLLNSVLEPINQALTLM (SEQ ID NO: 11);EPINQALTLMTKNVKPL (SEQ ID NO: 12); FAGVVLAGVALGVATAA (SEQ ID NO: 13);GVALGVATAAQITAGIA (SEQ ID NO: 14); TAAQITAGIALHQSNLN (SEQ ID NO: 15);GIALHQSNLNAQAIQSL (SEQ ID NO: 16); NLNAQAIQSLRTSLEQS (SEQ ID NO: 17);QSLRTSLEQSNKAIEEI (SEQ ID NO: 18); EQSNKAIEEIREATQET (SEQ ID NO: 19);TELLSIFGPSLRDPISA (SEQ ID NO: 20); PRYIATNGYLISNFDES (SEQ ID NO: 21);CIRGDTSSCARTLVSGT (SEQ ID NO: 22); DESSCVFVSESAICSQN (SEQ ID NO: 23);TSTIINQSPDKLLTFIA (SEQ ID NO: 24), SPDKLLTFIASDTCPLV (SEQ ID NO: 25) andSGRRQRRFAGVVLAGVA (SEQ ID NO: 26).

In a second aspect the present invention consists in a composition foruse in raising an immune response in an animal, the compositioncomprising at least one T helper cell epitope, the at least one T helpercell epitope being contained within a peptide sequence selected from thegroup consisting of SSKTQTHTQQDRPPQPS (SEQ ID NO: 1); QPSTELEETRTSRARHS(SEQ ID NO: 2); RHSTTSAQRSTHYDPRT (SEQ ID NO: 3); PRTSDRPVSYTMNRTRS (SEQID NO: 4); TRSRKQTSHRLKNIPVH (SEQ ID NO: 5); SHQYLVIKLIPNASLIE (SEQ IDNO: 6); IGTDNVHYKIMTRPSHQ (SEQ ID NO: 7); YKIMTRPSHQYLVIKLI (SEQ ID NO:8); KLIPNASLIENCTKAEL (SEQ ID NO: 9); AELGEYEKLLNSVLEPI (SEQ ID NO: 10);KLLNSVLEPINQALTLM (SEQ ID NO: 11); EPINQALTLMTKNVKPL (SEQ ID NO: 12);FAGVVLAGVALGVATAA (SEQ ID NO: 13); GVALGVATAAQITAGIA (SEQ ID NO: 14);TAAQITAGIALHQSNLN (SEQ ID NO: 15); GIALHQSNLNAQAIQSL (SEQ ID NO: 16);NLNAQAIQSLRTSLEQS (SEQ ID NO: 17); QSLRTSLEQSNKAIEEI (SEQ ID NO: 18);EQSNKAIEEIREATQET (SEQ ID NO: 19); TELLSIFGPSLRDPISA (SEQ ID NO: 20);PRYIATNGYLISNFDES (SEQ ID NO: 21); CIRGDTSSCARTLVSGT (SEQ ID NO: 22);DESSCVFVSESAICSQN (SEQ ID NO: 23); TSTIINQSPDKLLTFIA (SEQ ID NO: 24),SPDKLLTFIASDTCPLV (SEQ ID NO: 25) and SGRRQRRFAGVVLAGVA (SEQ ID NO: 26).

In a preferred embodiment of the present invention the compositioncomprises at least one peptide selected from the group consisting ofSSKTQTHTQQDRPPQPS (SEQ ID NO: 1); QPSTELEETRTSRARHS (SEQ ID NO: 2);RHSTTSAQRSTHYDPRT (SEQ ID NO: 3); PRTSDRPVSYTMNRTRS (SEQ ID NO: 4);TRSRKQTSHRLKNIPVH (SEQ ID NO: 5); SHQYLVIKLIPNASLIE (SEQ ID NO: 6);IGTDNVHYKIMTRPSHQ (SEQ ID NO: 7); YKIMTRPSHQYLVIKLI (SEQ ID NO: 8);KLIPNASLIENCTKAEL (SEQ ID NO: 9); AELGEYEKLLNSVLEPI (SEQ ID NO: 10);KLLNSVLEPINQALTLM (SEQ ID NO: 11); EPINQALTLMTKNVKPL (SEQ ID NO: 12);FAGVVLAGVALGVATAA (SEQ ID NO: 13); GVALGVATAAQITAGIA (SEQ ID NO: 14);TAAQITAGIALHQSNLN (SEQ ID NO: 15); GIALHQSNLNAQAIQSL (SEQ ID NO: 16);NLNAQAIQSLRTSLEQS (SEQ ID NO: 17); QSLRTSLEQSNKAIEEI (SEQ ID NO: 18);EQSNKAIEEIREATQET (SEQ ID NO: 19); TELLSIFGPSLRDPISA (SEQ ID NO: 20);PRYIATNGYLISNFDES (SEQ ID NO: 21); CIRGDTSSCARTLVSGT (SEQ ID NO: 22);DESSCVFVSESAICSQN (SEQ ID NO: 23); TSTIINQSPDKLLTFIA (SEQ ID NO: 24),SPDKLLTFIASDTCPLV (SEQ ID NO: 25) and SGRRQRRFAGVVLAGVA (SEQ ID NO: 26).

It is further preferred that the composition further comprises at leastone B cell epitope and/or at least one CTL epitope.

In yet another preferred embodiment the at least one B cell epitopeand/or the at least one CTL epitope are linked to at least one of the Thelper cell epitopes. It is also preferred that the compositioncomprises a plurality of epitope constructs in which each comprises atleast one T helper cell epitope and at least one B cell epitope.Alternatively the composition may comprises a plurality of epitopeconstructs in which each comprises at least one T helper cell epitopeand at least one CTL epitope.

It will be understood that the B cell epitope or CTL epitope may be anyepitope. A currently preferred B cell epitope is an LKRH B cell epitope.

The composition of the present invention may comprises a plurality of Thelper cell epitopes. These epitopes may be singular or be linkedtogether to form a single polypeptide. It will be understood that wherethe epitopes are linked to together in a single polypeptide the epitopesmay be contiguous or spaced apart by additional amino acids which arenot themselves part of the T helper cell epitopes.

As discussed above in one embodiment the T helper cell epitopes and atleast one B cell epitope and/or at least one CTL epitope in which theepitopes are linked. This may be done by simple covalent linkage of thepeptides. In another embodiment the epitopes are polymerised, mostpreferably such as described in PCT/AU98/00076, the disclosure of whichis incorporated herein by reference.

In yet another preferred embodiment the composition further comprises apharmaceutically acceptable excipient, preferably an adjuvant.

In a further aspect the present invention consists in a method ofinducing an immune response in an animal, the method comprisingadministering to the animal the composition of the second aspect of thepresent invention.

Pharmaceutically acceptable carriers or diluents include those used incompositions suitable for oral, rectal, nasal, topical (including buccaland sublingual), vaginal, parenteral (including subcutaneous,intramuscular, intravenous, intradermal, intrathecal and epidural)administration. They are non-toxic to recipients at the dosages andconcentrations employed. Representative examples of pharmaceuticallyacceptable carriers or diluents include, but are not limited to water,isotonic solutions which are preferably buffered at a physiological pH(such as phosphate-buffered saline or Tris-buffered saline) and can alsocontain one or more of, mannitol, lactose, trehalose, dextrose,glycerol, ethanol or polypeptides (such as human serum albumin). Thecompositions may conveniently be presented in unit dosage form and maybe prepared by any of the methods well known in the art of pharmacy.

As mentioned it is preferred that the composition includes an adjuvant.As will be understood an “adjuvant” means a composition comprised of oneor more substances that enhances the immunogenicity and efficacy of avaccine composition. Non-limiting examples of suitable adjuvants includesqualane and squalene (or other oils of animal origin); blockcopolymers; detergents such as Tween®-80; Quil® A, mineral oils such asDrakeol or Marcol, vegetable oils such as peanut oil;Corynebacterium-derived adjuvants such as Corynebacterium parvum;Propionibacterium-derived adjuvants such as Propionibacterium acne;Mycobacterium bovis (Bacille Calmette and Guerin or BCG); interleukinssuch as interleukin 2 and interleukin 12; monokines such as interleukin1; tumour necrosis factor; interferons such as gamma interferon;combinations such as saponin-aluminium hydroxide or Quil-A aluminiumhydroxide; liposomes; ISCOM adjuvant; mycobacterial cell wall extract;synthetic glycopeptides such as muramyl dipeptides or other derivatives;Avridine; Lipid A derivatives; dextran sulfate; DEAE-Dextran or withaluminium phosphate; carboxypolymethylene such as Carbopol′ EMA; acryliccopolymer emulsions such as Neocryl A640 (e.g. U.S. Pat. No. 5,047,238);vaccinia or animal poxvirus proteins; sub-viral particle adjuvants suchas cholera toxin, or mixtures thereof.

As will be recognised by those skilled in the art modifications may bemade to the peptides of the present invention without completeabrogation of biological activity. These modifications includeadditions, deletions and substitutions, in particular conservativesubstitutions. It is intended that peptides including such modificationswhich do not result in complete loss of activity as T helper cellepitopes are within the scope of the present invention.

Whilst the concept of substitution is well known in the field the typesof substitutions envisaged are set out below.

Original Residue Exemplary Substitutions Preferred Substitutions Ala (A)val; leu; ile Val Arg (R) lys; gln; asn Lys Asn (N) gln; his; lys; argGln Asp (D) glu Glu Cys (C) ser Ser Gln (Q) asn asn Glu (E) asp asp Gly(G) pro pro His (H) asn; gln; lys; arg arg Ile (I) leu; val; met; ala;phe, norleucine leu Leu (L) norleucine, ile; val; met; ala; phe ile Lys(K) arg; gln; asn arg Met (M) leu; phe; ile; leu Phe (F) leu; val; ile;ala leu Pro (P) Gly gly Ser (S) Thr thr Thr (T Ser ser Trp (W) Tyr tyrTyr (Y) trp; phe; thr; ser phe Val (V) ile; leu; met; phe ala;norleucine leu

Another type of modification of the peptides envisaged include, but arenot limited to, modifications to side chains, incorporation of unnaturalamino acids and/or their derivatives during peptide synthesis and theuse of crosslinkers and other methods which impose conformationalconstraints on the peptides.

Examples of side chain modifications contemplated by the presentinvention include, but are not limited to, modifications of amino groupssuch as by reductive alkylation by reaction with an aldehyde followed byreduction with NaBH₄; amidation with methylacetimidate; acylation withacetic anhydride; carbamoylation of amino groups with cyanate;trinitrobenzylation of amino groups with 2,4,6-trinitrobenzene sulphonicacid (TNBS); acylation of amino groups with succinic anhydride andtetrahydrophthalic anhydride; and pyridoxylation of lysine withpyridoxal-5′-phosphate followed by reduction with NaBH₄.

The guanidine group of arginine residues may be modified by theformation of heterocyclic condensation products with reagents such as2,3-butanedione, phenylglyoxal and glyoxal.

The carboxyl group may be modified by carbodiimide activation viaO-acylisourea formation followed by subsequent derivitisation, forexample, to a corresponding amide.

Tryptophan residues may be modified by, for example, oxidation withN-bromosuccinimide or alkylation of the indole ring with2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides. Tyrosine residueson the other hand, may be altered by nitration with tetranitromethane toform 3-nitrotyrosine derivative.

Modification of the imidazole ring of a histidine residue may beaccomplished by alkylation with iodoacetic acid derivatives orN-carbethoxylation with diethylpyrocarbonate.

Examples of incorporating unnatural amino acids and derivatives duringpeptide synthesis include, but are not limited to, use of norleucine,4-amino butyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid,6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine,ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid;2-thienyl alanine and/or D-isomers of amino acids.

The peptides of the present invention may be derived from CDV.Alternatively, the peptide or combination of peptide epitopes may beproduced by recombinant DNA technology. It is, however, preferred thatthe peptides are produced synthetically using methods well known in thefield. For example, the peptides may be synthesised using solutionsynthesis or solid phase synthesis as described, for example, in Chapter9 entitled “Peptide Synthesis” by Atherton and Sheppard which isincluded in a publication entitled “Synthetic Vaccines” edited byNicholson and published by Blackwell Scientific Publications. Preferablya solid phase support is utilised which may be polystyrene gel beadswherein the polystyrene may be cross-linked with a small proportion ofdivinylbenzene (e.g. 1%) which is further swollen by lipophilic solventssuch as dichloromethane or more polar solvents such as dimethylformamide(DMF). The polystyrene may be functionalised with chloromethyl oraminomethyl groups. Alternatively, cross-linked and functionalisedpolydimethyl-acrylamide gel is used which may be highly solvated andswollen by DMF and other dipolar aprotic solvents. Other supports can beutilised based on polyethylene glycol which is usually grafted orotherwise attached to the surface of inert polystyrene beads. In apreferred form, use may be made of commercial solid supports or resinswhich are selected from PAL-PEG-PS, PAC-PEG-PS, KA, KR or TGR.

In solid state synthesis, use is made of reversible blocking groupswhich have the dual function of masking unwanted reactivity in theα-amino, carboxy or side chain functional groups and of destroying thedipolar character of amino acids and peptides which render theminactive. Such functional groups can be selected from t-butyl esters ofthe structure RCO—OCMe₃—CO. Use may also be made of the correspondingbenzyl esters having the structure RCO—OCH₂—C₆H₅ and urethanes havingthe structure C₆H₅CH₂OCO—NHR which are known as the benzyloxycarbonyl orZ-derivatives and any Me₃—COCO—NHR, which are known as t-butoxylcarbonyl, or Boc derivatives. Use may also be made of derivatives offluorenyl methanol and especially the fluorenyl-methoxy carbonyl or Fmocgroup. Each of these types of protecting group is capable of independentcleavage in the presence of one other so that frequent use is made, forexample, of BOC-benzyl and Fmoc-tertiary butyl protection strategies.

Reference also should be made to a condensing agent to link the aminoand carboxy groups of protected amino acids or peptides. This may bedone by activating the carboxy group so that it reacts spontaneouslywith a free primary or secondary amine. Activated esters such as thosederived from p-nitrophenol and pentafluorophenol may be used for thispurpose. Their reactivity may be increased by addition of catalysts suchas 1-hydroxybenzotriazole. Esters of triazine DHBT (as discussed on page215–216 of the abovementioned Nicholson reference) also may be used.Other acylating species are formed in situ by treatment of thecarboxylic acid (i.e. the N-alpha-protected amino acid or peptide) witha condensing reagent and are reacted immediately with the aminocomponent (the carboxy or C-protected amino acid or peptide).Dicyclohexylcarbodiimide, the BOP reagent (referred to on page 216 ofthe Nicholson reference), O'Benzotriazole-N,N,N′N′-tetra methyl-uroniumhexafluorophosphate (HBTU) and its analogous tetrafluoroborate arefrequently used condensing agents.

The attachment of the first amino acid to the solid phase support may becarried out using BOC-amino acids in any suitable manner. In one methodBOC amino acids are attached to chloromethyl resin by warming thetriethyl ammonium salts with the resin. Fmoc-amino acids may be coupledto the p-alkoxybenzyl alcohol resin in similar manner. Alternatively,use may be made of various linkage agents or “handles” to join the firstamino acid to the resin. In this regard, p-hydroxymethyl phenylaceticacid linked to aminomethyl polystyrene may be used for this purpose.

Throughout this specification the word “comprise”, or variations such as“comprises” or “comprising”, will be understood to imply the inclusionof a stated element, integer or step, or group of elements, integers orsteps, but not the exclusion of any other element, integer or step, orgroup of elements, integers or steps.

DETAILED DESCRIPTION OF THE INVENTION

In order that the nature of the present invention may be more readilyunderstood preferred forms there of will now be described with referenceto the following non-limiting examples.

FIGURE LEGENDS

FIG. 1. Amino acid sequence of the fusion protein of CDV (SEQ ID NO: 27)

FIG. 2 a. Stimulation indices to Th-epitope P25 and its truncatedversions in Dog #70 immunised with P25-LHRH (X-axis concentration ofpeptides nmoles/well).

FIG. 2 b Stimulation indices to Th-epitope P25 and its truncatedversions in Dog #73 immunised with P25-LHRH (X-axis concentration ofpeptides nmoles/well).

FIG. 2 c Stimulation indices to Th-epitope P25 and its truncatedversions in Dog #127 immunised with P25-LHRH (X-axis concentration ofpeptides nmoles/well).

FIG. 2 d Stimulation indices to Th-epitope P25 and its truncatedversions in Dog #993 immunised with P25-LHRH (X-axis concentration ofpeptides nmoles/well).

FIG. 3 a. Stimulation indices to Th-epitope P27 and its truncated 15-merin Dog #105 immunised with P27-LHRH. (X-axis concentration of peptidesnmoles/well).

FIG. 3 b. Stimulation indices to Th-epitope P27 and its truncated 15-merin) Dog #94 immunised with P27-LHRH. (X-axis concentration of peptidesnmoles/well).

FIG. 3 c. Stimulation indices to Th-epitope P27 and its truncated 15-merin Dog #20 immunised with P27-LHRH. (X-axis concentration of peptidesnmoles/well).

FIG. 3 d. Stimulation indices to Th-epitope P27 and its truncated 15-merin Dog #101 immunised with P27-LHRH. (X-axis concentration of peptidesnmoles/well).

FIG. 4 a. Stimulation indices to Th-epitope P35 and its truncatedversions in Dog #19 immunised with P35-LHRH (X-axis concentration ofpeptides nmoles/well).

FIG. 4 b. Stimulation indices to Th-epitope P35 and its truncatedversions in Dog #100 immunised with P35-LHRH (X-axis concentration ofpeptides nmoles/well).

FIG. 4 c. Stimulation indices to Th-epitope P35 and its truncatedversions in Dog #96 immunised with P35-LHRH (X-axis concentration ofpeptides nmoles/well).

FIG. 4 d. Stimulation indices to Th-epitope P35 and its truncatedversions in Dog #102 immunised with P35-LHRH (X-axis concentration ofpeptides nmoles/well).

EXAMPLE 1

Identification of T Helper Cell Epitopes

Methods and Results:

Towards identification of canine T cell epitopes 94, 17 residueoverlapping peptides were designed encompassing the entire sequence offusion protein of canine distemper virus (CDV). The 17mer peptides werenumbered sequentially for identification starting from the N-terminus.The sequence of the fusion protein of CDV as determined by Barrett et al1987 (Virus Res. 8, 373–386) is shown in FIG. 1. The peptides were usedin T-cell proliferation assays using peripheral blood lymphocytes (PBMC)from dogs immunised with Canvac™ 3 in 1 vaccine (CSL Limited) whichcontains live CDV.

Initially, four dogs were used and they were boosted with the Canvac™ 3in 1 vaccine twice with four to six weeks between each vaccination. Thedogs were bled after each booster vaccination and the PBMCs were testedagainst the peptides. No significant proliferation to peptides wasobserved.

Since CDV has been reported to be lymphotropic and the vaccine consistsof live CDV, there was the possibility that it may be sequestered inlymphoid organs preventing significant numbers of precursor T cellsentering the peripheral system. To increase the frequency of peripheralblood anti-CDV T cells dogs were boosted with heat killed CDV (obtainedas a pellet from virus culture medium, CSL Limited). Two weeks later,the dogs were bled and the PBMCs tested for proliferation against thepeptides. Again there was no proliferation to the peptide antigens.

An alternate strategy was used to increase the precursor frequency ofspecific T cells recognising the CDV peptides. Fresh PBMCs obtained fromthese hyperimmunised dogs were subjected to stimulation in vitro withpools of all 94 peptides for 30 minutes at 37° C. The cells were thenwashed to remove any excess peptides and cultured for 7 days. Thispopulation of T cells was then tested with autologous APCs with everysingle peptide as the antigen. Table 1 shows the peptides to whichsignificant (stimulation index >2) levels of proliferation wereobserved.

To confirm this observation, the same four dogs were bled again, fiveweeks after receiving the dose of killed virus. The PBMCs werestimulated in vitro with pools of either all 94 peptides or peptides21–40 (because most of the activity was in this region) and after 7 daysof culture the stimulated T cells were tested against individualpeptides. Significant stimulatory indices were obtained with allpeptides, confirming the above results. Four more dogs which receivedonly one dose of 3 in 1 vaccine were tested using the in vitrostimulation method and all four dogs responded to the majority ofpeptides shown in Table 2.

The above peptides were also tested on cells from additional dogs, withresults shown in Table 3. Peptides P64, P74 and P75 were also shown toreact strongly with peripheral blood mononuclear cells from dogs ofvarious breeds immunised with CDV (Table 4), and are thereforeidentified as strong T-helper epitopes.

TABLE 1 Identification of canine T cell epitopes from the sequence offusion protein of CDV. Beagle Beagle Beagle Beagle Foxhound FoxhoundFoxhound Foxhound Peptides (Dog #18) (Dog #19) (Dog #20) (Dog #21) p2  2* <2 8 3.9 p4   4.9 <2 3.3 4.6 p6   2.5 <2 4 5.1 p10   2.3 <2 3.2 9.1p24   5.8 9.9 2.8 29 p25   3.2 11.9 4.5 17 p27   3.3 34 6.7 14.8 p29  3.5 42 4.4 <2 p35   3.1 57 3.3 22 p36   6.7 3.7 3.3 16 p37   6.9 10.98.2 26 p38   2.8 6.7 3.6 4.2 p47   3.3 85.7 2.9 1.9 p62 <2 51 5.6 4.2p68   6.6 <2 <2 11.7 *Stimulatory index

TABLE 2 Identification of canine T cell epitopes from the sequence offusion protein of CDV. Beagle Beagle Beagle Foxhound Foxhound FoxhoundBeagle Foxhound (Dog Peptides (Dog #70) (Dog #71) (Dog #72) #73) p8 2.2p22 2.6 p24 3.2 2.2 p25 1.5 2.9 2 12 p27 2.7 3.5 4.8 p28 2 p29 2 6 p331.6 p35 1.7 6.8 p37 1.7 p62 3

TABLE 3 Identification of canine T cell epitopes from the sequence offusion protein of CDV. Peptides Kelpie Foxhound (Dog#125) KelpieFoxhound (Dog#126) p23 3.2 p27 4.5 8.5 p28 1.9 p29 3.6 p33 6 p34 2.1 p353.8 10 p36 3 p37 2.5 p38 2.2 p39 2.9 p47 2.7 p62 2.4 p68 2.9

TABLE 4 Identification of canine T cell epitopes from the sequence offusion protein of CDV. Beagle Beagle Beagle Beagle Poodle Fox- Fox- Fox-Fox- Peptides Shitzu hound #18 hound #19 hound #20 hound #21 P64 50.02.5 2.5 P74 4.0 1.7 6.0 P75 10 2.5 7.2

Once again the same peptides and one additional peptide P32 were testedon cells from additional dogs. These peptides were also shown to reactstrongly with peripheral blood mononuclear cells from dogs of variousbreeds immunised with CDV (Table 5), and are therefore identified asstrong T-helper epitopes.

In conclusion, 26 peptides were identified as canine T helper cellepitopes in the fusion protein of CDV. The sequences of each of thesepeptides are set out in Table 6.

These T helper cell epitopes will have usefulness as components ofanimal, in particular, canine vaccines, either simply as syntheticpeptide based vaccines and as additions to vaccines containing morecomplex antigens.

TABLE 5 Identification of canine T cell epitopes from the sequence offusion protein of CDV. Poodle Grey Fox Terrier Kelpie Border PeptidesShitzu hound Terrier Cross Pointer Collie P2 140 <2 <2 <2 2.6 2 P4 44 2<2 2 3.5 2 P6 38 <2 <2 <2 <2 2 P8 100 2 <2 <2 2.8 2 P10 50 2 2.2 2.1 2.43 P25 <2 <2 2.6 <2 2.6 <2 P29 2 <2 <2 2 <2 <2 P32 <2 2 <2 <2 <2 <2 P33<2 <2 <2 <2 2 2 P35 <2 <2 2.2 <2 2 2 P37 <2 <2 <2 2 2 <2 P62 24 <2 <2 <2<2 <2 P64 50 <2 <2 <2 <2 <2 P68 5 <2 <2 <2 <2 <2 P74 4 <2 <2 <2 <2 <2P75 10 <2 <2 <2 <2 <2

TABLE 6 Sequences of the peptides: P2 SSKTQTHTQQDRPPQPS (SEQ ID NO: 1)P4 QPSTELEETRTSRARHS (SEQ ID NO: 2) P6 RHSTTSAQRSTHYDPRT (SEQ ID NO: 3)P8 PRTSDRPVSYTMNRTRS (SEQ ID NO: 4) P10 TRSRKQTSHRLKNIPVH (SEQ ID NO: 5)P24 SHQYLVIKLIPNASLIE (SEQ ID NO: 6) P22 IGTDNVHYKIMTRPSHQ (SEQ ID NO:7) P23 YKIMTRPSHQYLVIKLI (SEQ ID NO: 8) P25 KLIPNASLIENCTKAEL (SEQ IDNO: 9) P27 AELGEYEKLLNSVLEPI (SEQ ID NO: 10) P28 KLLNSVLEPINQALTLM (SEQID NO: 11) P29 EPINQALTLMTKNVKPL (SEQ ID NO: 12) P32 SGRRQRRFAGVVLAGVA(SEQ ID NO: 26) P33 FAGVVLAGVALGVATAA (SEQ ID NO: 13) P34GVALGVATAAQITAGIA (SEQ ID NO: 14) P35 TAAQITAGIALHQSNLN (SEQ ID NO: 15)P36 GIALHQSNLNAQAIQSL (SEQ ID NO: 16) P37 NLNAQAIQSLRTSLEQS (SEQ ID NO:17) P38 QSLRTSLEQSNKAIEEI (SEQ ID NO: 18) P39 EQSNKAIEEIREATQET (SEQ IDNO: 19) P47 TELLSIFGPSLRDPISA (SEQ ID NO: 20) P62 PRYIATNGYLISNFDES (SEQID NO: 21) P68 CIRGDTSSCARTLVSGT (SEQ ID NO: 22) P64 DESSCVFVSESAICSQN(SEQ ID NO: 23) P74 TSTIINQSPDKLLTFIA (SEQ ID NO: 24) P75SPDKLLTFIASDTCPLV (SEQ ID NO: 25)

Selected sequences of the identified T-cell epitopes were tested fortheir ability to induce an antibody response to a linked B-cell epitope.Trials were conducted in dogs for assessment of antibody responses. TheT-cell epitopes were linked to the B cell epitope LHRH (leuteinisinghormone releasing hormone), with the T-cell epitope at the N-terminusand LHRH positioned at the carboxy terminus.

Peptides were synthesised using standard chemistry with Fmoc protection.All peptides were purified to at least 80% purity and the productchecked by mass spectroscopy.

The peptides were produced as contiguous T-cell—B cell determinants. TheLHRH sequence of Pyro Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly (SEQ IDNO: 28), or variations of it, was linked to the carboxyl terminus ofeach respective CDV T-helper epitope.

In-vivo evaluation of some of the T-helper epitopes was conducted in twotrials, by vaccination of dogs with T-helper—LHRH sequences.

EXAMPLE 2 Trial K9-5

A total of 14 dogs of mixed sex were used in this trial. All had beenpreviously vaccinated with a live CDV vaccine and had also beenvaccinated against LHRH. Vaccine formulation.

Test peptides P25, P27, P35 from CDV were synthesised with LHRH at the Cterminus of each T-helper epitope. The LHRH sequence used was the full10 amino acids of the native LHRH. Each of the vaccine constructs,together with a control peptide comprising a mouse influenza T-cellepitope linked to a repeat malarial B-cell epitope (sequence shown intable below) were purified to ˜80–90% purity. All peptides weredissolved in 4M urea before dilution with sterile saline to anappropriate volume to give 40 nmoles per 1 mL dose. Iscomatrix™ wasadded to a final concentration of 150 ug/1 mL dose as adjuvant togetherwith thiomersal preservative (0.01%).

ISCOM™ or Immunostimulating Complex (Barr, Sjolander and Cox, 1998,Advanced Drug Delivery Systems 32: 247–271) are a well characterisedclass of adjuvant comprised of a complex of phospholipid, cholesteroland saponin, usually with a protein incorporated into the complex. Wherethe complex is formed in the absence of protein antigen, then thiscomplex is termed Iscomatrix™. The saponin used in the preparation ofthis adjuvant was Quil A.

Vaccination, Blood Samples and Assays.

All dogs were vaccinated with a 1 mL dose, delivered in the scruff ofthe neck. Vaccinations were given at 0 and 4 weeks and venous bloodsamples were obtained at intervals during the trial.

Effective T-cell help was determined by measuring the antibody responseto LHRH by ELISA. Biological effectiveness of the peptide based vaccinewas determined by measuring the levels of progesterone in female dogsand testosterone in male dogs.

TABLE 7 Trial Groups Peptide Dog Nos. Control-ALNNRFQIKGVELKS-(NANP)3104, 998 (SEQ ID NO: 30) P25-LHRH 1–10 70, 73, 127, 993 P27-LHRH 1–1020, 94, 101, 105 P35-LHRH 1–10 19, 96, 100, 102Results

Pre-existing low antibody levels to LHRH were present in all dogs due toimmunisation previously with a different vaccine. The control group ofdogs exhibited a slow decrease in antibody levels.

Dogs immunised with P25-LHRH, P27-LHRH and P35-LHRH all showed strongantibody responses to the B-cell epitope (LHRH). This response persistedto 6 weeks post boost vaccination (see Table 8).

The biological potency of the vaccine was demonstrated by a significantreduction in progesterone or testosterone levels (see Tables 9 and 10).

TABLE 8 Anti LHRH Titres Anti LHRH Titres Dog 2 wks 6 wks Peptide NoPrebleeds post boost post boost Control 104 1258  1975  1936 998 2559 1982  1947 Average 1794  1978  1941 Range 1258–2559 1975–1982 1936–1947P25-LHRH 70 856 24245 16697 (1–10) 73 42665 16922 127 1361 21485 19662993 577 24879 15119 Average 886 23120 17242 Range   0–1361 21485–4266515119–19662 p27-LHRH 20 747 29653  8423 (1–10) 94 41247 22759 101 425652724 17353 105 944 12600  8366 Average 2004 25774 12049 Range  747–425612600–52724 8366–22759 p35-LHRH 19 665 18033  6228 (1–10) 96 1621 26583 5744 100 580 17255  4829 102 180 11740  2963 Average 323 14233  3783Range  180–1621 11740–26583 2963–6228

TABLE 9 Progesterone results (nmol/L) Dog 4 wks 2 wks 6 wks Peptide No.post primary post boost post boost Control 998 5.17 4.28 <0 p25-LHRH(1–10) 127 3.04 4.83 <0 993 1.7 0.87 <0 p27-LHRH (1–10) 101 0.42 0.14 <0p35-LHRH (1–10) 96 31.76 2.15 <0 100 <0 <0 <0

TABLE 10 Testosterone results (nmol/L) Dog 4 wks 2 wks 6 wks Peptide No.post primary post boost post boost Control 104 9.69 2.51 3.31 p25-LHRH(1–10) 70 <0 <0 <0 73 5.38 <0 <0 p27-LHRH (1–10) 20 1.04 <0 <0 94 3.33<0 <0 105 >47.7 <0 <0 p35-LHRH (1–10) 19 4.3 2.77 4.55 102 6.72 <0 <0

The effectiveness of selected T-cell epitopes from the F-protein of CDVin providing T-cell help to elicit antibody responses in dogs provesthat the identified sequences are functional. These results alsovalidate the scientific approach and usefulness of the in vitroscreening method for identifying T-helper epitope sequences with in vivoactivity.

EXAMPLE 3 Trial K9-8

A total of 35 dogs mixed sex were used in this trial. All had beenpreviously vaccinated with a live CDV vaccine but had not beenvaccinated against LHRH.

Vaccine Formulation:

The T-Helper epitopes were linked to a truncated form of LHRH,containing amino acids 2 to 10 of the native 10 amino acid sequence, asshown below:

2–10 LHRH His - Trp - Ser - Tyr - Gly - Leu - Arg - Pro - Gly. (SEQ IDNO: 29)

All vaccines were formulated as for Example 2, ie each 1 mL dose ofvaccine contained 40 nmoles of peptide, 150 μg Iscomatrix™, andthiomersal as preservative.

Where dogs were vaccinated with a pool of peptides, the concentration ofeach peptide was adjusted to give equal concentrations and a totalamount of 40 nmoles of LHRH epitope per 1 mL dose.

Vaccination, Blood Samples and Assays.

All dogs were vaccinated with a 1 mL dose, delivered in the scruff ofthe neck. Vaccinations were given at 0 and 4 weeks and venous bloodsamples were obtained at intervals during the trial.

Effective T-cell help was determined by measuring the antibody responseto LHRH by ELISA. Biological effectiveness of the peptide based vaccinewas determined by measuring the levels of progesterone in female dogsand testosterone in male dogs.

TABLE 11 Trial Groups Peptide Group Dog Nos. P25-LHRH 2–10 211, 195,197, 181 P27-LHRH 2–10 203, 191, 186, 201 P35-LHRH 2–10 217, 198, 187,196 Pool: P25-LHRH 2–10, 212, 193, 178, 216, Y3 P27-LHRH 2–10, P35-LHRH2–10 P2-LHRH 2–10 194, 199, 179, 220 P8-LHRH 2–10 Y4, Y6, 160, 200P62-LHRH 2–10 219, 185, 221, 177 P75-LHRH 2–10 189, 222, 202, 176Unvaccinated controls 190, 159Results

Strong antibody responses to LHRH were demonstrated in dogs immunisedwith the T-cell-LHRH constructs with the T-cell epitopes P25, P27, P35,P62, P75, and the pool of T-cell-LHRH peptides comprising a combinationof T-cell epitopes P25, P27 and P35 (see Table 12).

Low to undetectable antibody responses were seen in dogs immunised withP2 and P8-LHRH peptides (see Table 12). This was concluded to indicatethat these T-cell peptides were not well recognised by Beagle-Foxhounddogs, which is consistent with their identification using PBMCs' fromother dog breeds. The initial screening in Beagle foxhound dogsindicated that this breed of dog does not respond to these 2 T-cellepitopes.

As is well understood by those skilled in the art of peptide vaccinesthe response to individual peptides is genetically determined. The classII Major Histocompatability Complex (MHC II) is polymorphic. Class IImolecules at the cell surface function to bind peptides for presentationto T-cells, which is required as part of the activation process forT-cells, including helper T-cells. The allelic forms of MHC class IIbind discrete sets of peptide antigens, and thus the response to thoseantigens is genetically determined. Thus the results are interpreted toindicate that the Beagle—Foxhound breed of dog does not possess theappropriate MHC-II alleles to respond to P2 and P8, but that otherbreeds of dog do, eg. the Poodle Shitzu breed that were used to identifythese peptides.

Control dogs showed no change in antibody levels to LHRH during thetrial period and hormone levels were within normal ranges for the ageand sex of the dogs (see Table 12).

TABLE 12 Anti-LHRH Titres Anti LHRH Titres 4 wks after 2 wks post 4 wkspost Peptide Group Dog No primary boost boost Control 1 159 0 0 0 1 1900 0 0 GMT Pool 2 Y3 1860 55659 95038 2 178 17900 416036 486793 2 1938770 211369 189143 2 212 3766 121411 135293 2 216 8378 294769 642293 GMT6207 177292 237798 P25-LHRH 3 181 1893 152264 131643 3 195 31197 205906455193 3 197 14423 337698 240543 3 211 20607 142798 131643 GMT 11510193037 214229 P27-LHRH 4 186 0 11206 17263 4 191 0 59154 125493 4 201 017041 34103 4 203 0 1000 857 GMT 0 18523 26698 P35-LHRH 5 187 2009141775 55797 5 196 4868 237208 158040 5 198 1539 154375 68307 5 217 0121050 40822 GMT 2469 103085 58002 P2-LHRH 6 179 0 0 0 6 194 0 0 0 6 1990 0 0 6 220 0 0 0 GMT P8-LHRH 7 Y4 0 0 0 7 Y6 0 0 0 7 160 0 1200 ND 7200 0 8000 2227 GMT P62-LHRH 8 177 1242 3821 2985 8 185 0 146581 67461 8219 0 29353 28282 8 221 2697 231473 156549 GMT 1830 44167 30728 P75-LHRH9 176 0 12177 5559 9 189 0 15795 17155 9 202 0 2121 2216 9 222 0 97877879 GMT 11201 8746

EXAMPLE 4

In Vitro T Cell Proliferation Assays to Demonstrate Recognition ofTh-epitope incorporated in the Peptide Vaccines

To demonstrate recognition of the Th-epitope within the peptideimmunogen PBMCs obtained from dogs immunised with peptide vaccines (dogsfrom Example 2) were tested against the respective Th-epitopes. Theassay was carried out without the enrichment of PBMCs. PBMCs obtainedfrom Ficoll gradient purification were directly tested against therespective Th-epitope and its truncated versions. The study demonstratedthat all the dogs immunised with peptide vaccines responded to theTh-epitope incorporated confirming that T-cell activity resides in therespective sequences (FIGS. 2–4). Truncated versions of the respectiveTh-epitopes were also tested to more closely define the T-cell activitywithin the sequences. It was observed that for P25 the full sequence of17 residues was better than the shorter peptides of 15 and 12 residues,each truncated from the N-terminus of the sequence (FIG. 2). Thisimplies that the T-cell activity is towards the N-terminus or middle ofthe 17-residue peptide.

A similar observation was made with P27, the 17 residue long peptide wasa better simulator than the 15-mer truncated from the N-terminus (FIG.3). This observation again suggested that the T-cell activity may residetowards the middle or the N-terminus of the full length peptide.

In the case of P35 and its shorter versions, except for one dog (#102),the other three dogs responded as well to the 12 residue peptide as tothe full length 17 residue one (FIG. 4). In dog # 102 the 15 residuepeptide was more stimulatory than the full length peptide. From this itcan be deduced that that the first two residues in the sequence of P35may not be essential and that the activity is towards the middle orC-terminus of the peptide.

EXAMPLE 5

Trial in BALB/c Mice

The canine vaccines with CDV-F derived Th-epitopes and LHRH used inExample 3 were also used to immunise BALB/c mice to investigate if theTh-epitopes would be functional in a different animal species.

Vaccine Formulation

All vaccines were formulated as for Example 3 except that they werediluted further so that 100 μl doses contained 2.7 nmoles of peptide and10 μg of Iscomatrix™ and thiomersol as preservative.

Vaccination, Blood Samples and Assays.

Mice were vaccinated with 100 μl of the vaccine at the base of tail.Vaccinations were given at 0 and 4 weeks and animals bled at intervalsafter each vaccination from the retro-orbital plexus. Effective T-cellhelp was determined by measuring the antibody response to LHRH by ELISA.

Results

Mice immunised with P25-LHRH and pool of peptides comprising ofP25-LHRH, P27-LHRH and P35-LHRH generated high antibody titres to LHRH.Peptides P35 and P75 generated low antibody titres whereas miceimmunised P2, P8 and P62 had undetectable levels of anti-LHRH antibodies(Table 13).

It will be appreciated by persons skilled in the art that numerousvariations and/or modifications may be made to the invention as shown inthe specific embodiments without departing from the spirit or scope ofthe invention as broadly described. The present embodiments are,therefore, to be considered in all respects as illustrative and notrestrictive.

TABLE 13 Anti-LHRH antibody titres in mice immunised with CDV-F derivedT cell epitope-LHRH vaccines 4 weeks post first vaccination 2 weeks postsecond vaccination Groups Mouse 1 Mouse 2 Mouse 3 Mouse 4 Mouse 5 Mouse1 Mouse 2 Mouse 3 Mouse 4 Mouse 5 Group <100 <100 <100 <100 1(control)Group 2 100 126 200 126 200 16,000 16,000 16,000 16,000 16,000 (pool)Group 3 126 400 282 100 282 16,000 16,000 16,000 16,000 16,000(p25-LHRH) Group 5 <100 <100 <100 <100 <100 1,412 800 <100 <100 <100(p35-LHRH) Group 6 <100 <100 <100 <100 (p2-LHRH) Group 7 <100 <100 <100<100 <100 <100 <100 <100 (p8-LHRH Group 8 <100 <100 <100 126 126 <100(p62-LHRH Group 9 <100 <100 <100 <100 <100 <100 <100 316 3,162 <100(p75-LHRH

1. A T helper cell epitope, the epitope being contained within a peptidesequence selected from the group consisting of SSKTQTHTQQDRPPQPS (SEQ IDNO: 1); QPSTELEETRTSRARHS (SEQ ID NO: 2); QSLRTSLEQSNKAIEEI (SEQ ID NO:18); and DESSCVFVSESAICSQN (SEQ ID NO: 23).
 2. A composition for use inraising an immune response in an animal, the composition comprising atleast one T helper cell epitope, the at least one T helper cell epitopebeing contained within a peptide sequence selected from the groupconsisting of SSKTQTHTQQDRPPQPS (SEQ ID NO: 1); QPSTELEETRTSRARHS (SEQID NO: 2); QSLRTSLEQSNKAIEEI (SEQ ID NO: 18); and DESSCVFVSESAICSQN (SEQID NO: 23), and a pharmaceutically acceptable carrier.
 3. A compositionas claimed in claim 2 in which the composition comprises at least onepeptide selected from the group consisting of SSKTQTHTQQDRPPQPS (SEQ IDNO: 1); QPSTELEETRTSRARHS (SEQ ID NO: 2); QSLRTSLEQSNKAIEEI (SEQ ID NO:18); and DESSCVFVSESAICSQN (SEQ ID NO: 23).
 4. A composition as claimedin claim 2 in which the composition further comprises at least one Bcell epitope and/or at least one CTL epitope.
 5. A composition asclaimed in claim 4 in which the at least one B cell epitope and/or theat least one CTL epitope are linked to at least one of the T helper cellepitopes.
 6. A composition as claimed in claim 5 in which thecomposition comprises a plurality of epitope constructs in which eachconjugate comprises at least one T helper cell epitope and at least oneB cell epitope.
 7. A composition as claimed in claim 5 in which thecomposition comprises a plurality of epitope constructs in which eachconjugate comprises at least one T helper cell epitope and at least oneCTL epitope.
 8. A composition as claimed in claim 4 in which thecomposition comprises an LHRH B cell epitope.
 9. A composition asclaimed in claim 2 in which the composition comprises a plurality of Thelper cell epitopes.
 10. A composition as claimed in claim 9 in whichthe plurality of T cell epitopes is a single polypeptide.
 11. Acomposition as claimed in claim 9 in which the composition furthercomprises at least one B cell epitope and/or at least one CTL epitope.12. A composition as claimed in claim 11 in which the least one B cellepitope and/or at least one CTL epitope is linked to the plurality of Thelper cell epitopes.
 13. A composition as claimed in claim 11 in whichthe composition comprises an LHRH B cell epitope.
 14. A composition asclaimed in claim 2 in which the composition comprises an adjuvant.
 15. Acomposition as claimed in claim 14 in which the adjuvant comprisesISCOMs or Iscomatrix.
 16. A method of inducing an immune response in ananimal, the method comprising administering to the animal thecomposition as claimed in claim 2.